Fragment design
When starting from DNA oligos, the number of junctions needed to assemble a construct depends on the length of the construct and the length of the starting oligos being used. A list of all of the component oligos that compose the Sidewinder fragments used in these experiments is provided in Supplementary Table 1. For an oligo of length L, the maximum bases of coding information for a fragment composed from these oligos (L c ) is L − 2L b , where L b is the length of the barcode. Toeholds are then chosen starting maximally from L c bases away from the previous junction. Hand-designed assemblies standardly use the maximal length fragments and use toeholds from position L c –10 to L c but can be shifted to avoid unintended toehold secondary structure. NUPACK-designed assemblies choose a 10-base toehold within the range of position L c −25 to L c with an ensemble defect of <0.1 from a secondary-structure-free toehold.
We tested a range of toehold lengths and designs, varying the ligation site from −10 bases to +10 bases on either side of the Sidewinder helix. We found effective ligation occurring equal to or further than ±6 bases from the Sidewinder helix (Extended Data Fig. 2a,b). This led us to standardize the toehold length to 10 bases for the experiments described in this paper as to ensure sufficient distance of the nick from the Sidewinder helix to accommodate ligase docking and effective ligation.
Barcodes were designed to be compatible with their respective toehold after the location of the junction is chosen. Barcode sequences were chosen or generated on the basis of the predicted secondary structure and crosstalk between other toehold–barcode sequences at the assembly’s ligation temperature. The h-fibroin and parallel assembly barcodes were designed using a guess–check method, choosing from a set list of pregenerated orthogonal barcodes15. Starting with the first toehold, a barcode sequence is arbitrarily chosen (‘guess’) and appended to the 3′ end of the toehold and checked for secondary structure at 50 °C with complex size 2 using the NUPACK web browser16,17 (‘check’). The subsequent barcodes are then chosen from the pregenerated list, checked individually in the same manner, then checked for cross reactivity against all previously chosen toehold-barcode sense and antisense sequences at 50 °C and complex size 2. All barcodes in this study use natural bases but we anticipate that the specificity and diversity of Sidewinder barcodes can be expanded to include unnatural bases and other DNA nanotechnology interactions.
The 5-to-40-piece Lux assemblies, the APOE assembly and the library assembly had bespoke barcode sequences generated for the specific assembly using NUPACK Python package16. Target strand (NUPACK variable) secondary structure was defined to be fully unpaired for each single stranded barcode/toehold pair combination. Complexes (NUPACK variable) were defined to take on the desired 3WJ structure for barcode/toeholds. Step tubes (NUPACK variable) are defined such that, in step 0, individual barcode/toehold sequences take on the desired unpaired secondary structure before assembly; and, in step 1, barcode/toehold sequences pair with the intended assembly partner during assembly at 50 °C with an ensemble defect of <0.1. After barcode generation, all secondary structures and the cross-reactivity of chosen barcodes were checked using the NUPACK web browser16,17.
For the length of the Sidewinder barcodes, we tested barcode lengths from 15 to 21 bases, both with and without a T–T or U–U mismatch at the base of the 3WJ for added stability46 and neither seem to have bearing on ligation efficiency (Supplementary Table 1). We also tested a variety of commercially available ligases and found high variability in ligation efficiency at −10 bases from the Sidewinder helix across the ligases tested (Extended Data Fig. 2c). Of the various ligases tested, Taq ligase and HiFi Taq ligase were picked as the preferred ligases due to their efficiency of ligation at the 3WJ and stability at extremely high temperatures.
Oligo purchasing
All assembly oligos were purchased from Millipore-Sigma with standard DNA synthesis for DNA oligos in tubes, which has a max oligo length of 120 bases. The only exception was fragment 4 of the identical toehold assembly, which was ordered as a long oligo to enable four identical toeholds (Supplementary Table 1). Barcode oligos were ordered with standard desalt purification and coding oligos were ordered PAGE purified, but this has since been seen to be superfluous (Extended Data Fig. 3f). Both barcode and coding oligos for the fluorescent protein library were ordered with cartridge purification. Both barcode and coding oligos for the Sidewinder characterization in Fig. 1 were ordered PAGE purified. PCR amplification primers were ordered from Integrated DNA Technologies with standard desalt purity. All oligos are listed in Supplementary Table 1. All Sidewinder component oligos were shipped dry.
Heteroduplex annealing
Oligos are suspended by hand in 1× TE buffer at pH 8.0 (Corning, Thermo Fisher Scientific) to a final concentration of 100 μM on the basis of the manufacturer’s reported weight. To ensure adequate resuspension of the dried oligos, if the volume required to for a final concentration of 100 μM was less than 50 μl of TE buffer according to the manufacturer’s reported weight, the oligos would be resuspended in a volume of 50 μl of buffer, resulting in a lower final concertation. The concentration of all oligos was additionally measured using the Qubit ssDNA Assay Kit (Invitrogen, Thermo Fisher Scientific) and final concentration calculations were based on these measurements.
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